The TRPM2 Ca2+-permeable cation channel opens in response to binding of ADP-ribose to a well defined C-terminal gating domain. TRPM2 has been shown to be involved in oxidative-stress induced calcium entry, where sustained TRPM2 channel opening results from release of ADP-ribose by mitochondria as a 2nd messenger (47). A major question regarding TRPM2-dependent calcium signals is the nature of the role they play in oxidative-stress induced cellular responses. Addressing this type of question in primary cell types and in vivo animal models is of particular interest, as TRPM2 has been found in both striatal neurons and pancreatic beta cells, cell types which are known to be especially susceptible to influence or damage by oxidative stress. However, no animal models of TRPM2-deficiency are presently available, and no small molecules are presently available which are capable of specifically modulating oxidative-stress induced calcium signals mediated by TRPM2. The development of specific small molecule modulators of oxidative stress induced TRPM2 calcium signals would provide important tools for use in understanding TRPM2's role in the physiology and pathophysiology of oxidative stress, and has the potential to contribute to the development of clinically useful therapeutics for pathophysiologic situations in which oxidative stress-induced TRPM2 gating is implicated in cell damage. In the course of our work on TRPM2 function, my laboratory has developed an HEK-293 cell line (HEKTRPM2 cells) which constitutively expresses a high level of human TRPM2 channels, the biophysical properties of which are indistinguishable from those of native human TRPM2 (45). The HEK-TRPM2 cell line grows rapidly, has exhibited stable TRPM2 expression over many passages, and is adherent, properties compatible with its use in both fluorescent reporter primary HTS screens and medium-throughput patch clamp assays required for secondary screening, hit optimization and specificity screening. In this R21 grant, we propose to use the HEK-TRPM2 cell line to develop optimized assays for use in high throughput small molecule screens for modulators of TRPM2-mediated calcium signals. In Specific Aim 1, we will compare HEK-TRPM2 cell responses to fluorescent calcium indicators and membrane potential reporters to determine which readout is best suited to HTS screening for TRPM2 modulators. In Specific Aim 2, we will utilize the fluorescent reporter system identified in Specific Aim 1 and adapt its use with HEK-TRPM2 cells for 96 and 384 well plate format assays. Using the 96 well plate format, we will also evaluate the overall method's properties and suitability for HTS using a library of compounds of known bioactivity.